In accordance with rapid development of techniques in the field of biotechnology including molecular biology, various analytical techniques which help the research activities in this technical field have also been developed from day to day. Particularly, the separation, purification and identification of DNA on polymer gels are the essential key techniques in the field of biotechnology and their use and importance have been increased. The separation and identification of DNA have generally been accomplished by electrophoresis on polymer gels such as agarose gels or polyacrylamide gels. Since polymer gels can be prepared in various types and sizes and their porosity can also be varied, polymer gels are of wide application. Although the polyacrylamide gel has an excellent separating ability sufficient to distinguish even the difference of 1 bp, it is suitable for the separation of small fragment DNAs having 5-500 bp because it can be applied only within the narrow range. Contrary to this, the agarose gel has been widely used due to its applicable range as broad as 200 bp-50 kb DNAs.
To detect DNA fragments separated on polymer gels, DNA staining method using Ethidium bromide (hereinafter, abbreviated to EB) has been the most generally used [see Sharp, P. A., Sugden, B. and Sambrook, J., Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose-ethidium bromide electrophoresis, Biochemistry 12 (16), 3055-3063 (1973)]. EB has a planar aromatic ring-containing structure which can be intercalated into the helical structure of DNA, as represented by the following chemical formula (1) and is fixed between DNA bases to display an increased fluorescence upon radiation of ultraviolet ray: ##STR1##
The difference in fluorescences between EB combined to DNA and background EB allows to detect the DNA band and reaches about 40 times. The staining with EB is conducted by adding EB at a concentration of 0.5 .mu.g/ml to electrophoresis buffer solution and an agarose gel and, after completion of electrophoresis, mounting the gel on a transilluminator and then applying the radiation of ultraviolet ray to the gel to detect the DNA band. Alternatively, the gel obtained after electrophoresis can be stained by immersing the gel into EB solution (0.5 .mu.g/ml), washed with water and then mounted on a transilluminator to detect the DNA band [see Sambrook, J., Fritsch, E. F. and Maniatis, T.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor University Press (1989)].
Although the above-mentioned methods have been widely used because of their relatively simple procedure and high sensitivity (1-10 ng), they have serious problems as specifically described below. First, since EB is a potent mutagen, it must be substantially carefully handled and wasted. Second, since ultraviolet ray having short wavelength (254-310 nm) is radiated, the use of any shield for protection of experimenters from the radiation is required. Third, toxic substances such as ozone is produced by UV light; fourth, DNA damages such as DNA dimerization, nicking, bleaching, etc., may be caused upon UV radiation [see Brunk, C. F., Simson, L., Analytical Biochemistry 82, 455-462 (1977)]; and fifth, the photographing equipment is required to take photographs for obtaining the record of experimental result.
As the methods to improve the above-mentioned problems involved in EB staining method, silver staining method, method for detection of prestained DNA using confocal laser scanning fluorescence imaging system, staining method using imidazole and zinc, and staining method using a visible dye have been reported.
Although silver staining method has advantages that it has better sensitivity than EB and does not require UV radiation, its general use is limited due to very complicated procedure and high cost for purchasing reagents [see Datar, R. H. and Bhisey, A. N., A sensitive method for permanent silver staining of DNA in agarose gels, Indian Journal of Biochemistry & Biophysics 25, 373-375 (1988); Peats, S., Quantitation of protein and DNA in silver-stained agarose gels, Analytical Biochemistry 140, 178-182 (1984); Gottlieb, M. and Chavko, M., Silver staining of native and denatured eukaryotic DNA in agarose gels, Analytical Biochemistry 165, 33-37 (1987); and Beidler, J. L., Hilliard, P. R and Rill, R. L., Ultrasensitive staining of nucleic acids with silver, Analytical Biochemistry 126, 374-380 (1982)].
The method for detection of prestained DNA using confocal laser scanning fluorescence imaging system is conducted by subjecting DNA prestained with EB homodimer, oxazole yellow homodimer, thiazole orange homodimer, etc. to electrophoresis and then detecting the DNA by means of a confocal laser scanning fluorescence imaging system [see Ray, H. S., Quesada, M. A., Peck, K., Mathies, R. A. and Glazer, A. N., High-sensitivity two-color detection of double-stranded DNA with a confocal fluorescence gel scanner using ethidium homodimer and thiazole orange, Nucleic Acid Research 19 (2), 327-333 (1991)]. This method has the advantage of a very high sensitivity but has the disadvantage in that it requires the use of very expensive apparatus.
The staining method using imidazole and zinc ion is a method wherein DNA can be detected by combining Zn.sup.2+ to DNA and then adding imidazole thereto to produce an insoluble white precipitate on DNA band [see Hardy, E., Sosa, A. E., Pupo, E., Casalvilla, R. and Fernandez-Patron, C., Zinc-imidazole positive: A new method for DNA detection after electrophoresis on agarose gels not interfering with DNA biological integrity, Electrophoresis 17, 26-29 (1996)]. The other method using imidazole and zinc ion is a background staining method for DNA detection in which the precipitate of Zn2+-imidazole is produced on the gel surface except DNA band [see Hardy, E., Pupo, E., Casalvilla, R., Sosa, A. E., Trujillo, L. E., Lopez, E. and Castellanos-Serra, L., Negative staining with zinc-imidazole of gel electrophoresis-separated nucleic acids, Electrophoresis 17, 1537-1541 (1996)]. The Zn2+-imidazole methods have some advantages that they use other reagents than toxic substances such as EB and their sensitivities are not lower than that of EB. Furthermore, since they do not require the use of UV radiation, the problems caused by UV radiation, including DNA damages, do not occur and they are suitable for preparative purpose. However, the methods have also disadvantages in that they are conducted through relatively complicated detection procedures, the permanent deposit of samples is impossible (the sample can be stored for only 2 months in distilled water), and the contrast between DNA band and background is visibly unclear.
The staining with a visible dye is a method in which a dye having the structure similar to that of EB, such as Brilliant cresyl blue, Nile blue, Methylene blue, etc., is used and DNA can be detected only through staining and destaining procedures without ultraviolet ray radiation. However, this has some disadvantages in that the staining and destaining procedures need a long time and the sensitivity is low (15-40 ng). For example, using Methylene blue, it takes from 2 hours to a few days for destaining procedure. The sensitivity is 25 ng using Brilliant cresyl blue, 40 ng using Nile blue and 15-20 ng using Methylene blue, respectively [see Santillan-Torres, J. L. and Ponce-Noyola, P., A novel stain for DNA in agarose gels, Trend genet 9 (2), 40 (1993); and Adkins, S. and Burmeister, M., Visualization of DNA in agarose gels as migrating colored bands: Applications for preparative gels and educational demonstrations, Analytical Biochemistry 240, 17-23 (1996)].
In addition to the above-mentioned methods, the method for detection of DNA combined to biotin on agarose gels without blotting procedure has also been reported [see Sun, Y., Detection of biotinylated nucleic acids directly on agarose gels, Biotechniques 16 (5), 782-784 (1994)]. However, this is also conducted through complicated procedures.
In another aspect, Janus blue is a cationic dye having the structure containing a quaternary amine and a planar aromatic ring which can be intercalated into DNA helix. Literature [Dutt M K, Microsc Acta, 1982, 85 (4), 361-368] describes a method for staining cell nuclei from tissue section in which RNA has been selectively extracted with cold phosphate buffer solution, using an aqueous solution containing Janus blue, Methylene blue and Janus red. However, this differs in a good deal from the method for detection of DNA on polymer gels according to the present invention. Literature [Steve Adkins et al., Analytical Biochemistry 240, 17-23, 1996] describes that by inclusion of various common commercially available visible dyes (such as Crystal violet, Methyl green, Pyronin Y, Thionin, Basic blue 66, Basic red 29, Safranin O, Janus green B, Nile blue, Pinacyanol, Stains-all, Basic yellow 11, Alcian blue 8GX, and Ruthenium red) in standard agarose gels, DNA bands are observable in visible light as they are separating. According to the above literature, such bands could be directly recovered from gels (approximately 50% yield) and used in standard enzymatic reactions without purification and of common commercially available dyes, Nile blue gave the sharpest and most persistent bands. Additionally, they reported that bands containing greater than 40 ng DNA could be detected by direct visual inspection of gels during electrophoresis and drying the gels increased sensitivity to 4 ng. However, it differs from the present invention in staining method and has drawbacks in that it has low detection sensitivity.
Thus, the present inventors have extensively and repeatedly studied for improvement of the prior staining method with visible dye in order to develop the method for detection of DNA in a sensitivity comparable to that of EB staining method and in a rapid, simple and safe manner, which can be used as a substitute for the prior EB staining method involving the problems which may be caused by toxicity and mutagenicity of EB itself and by the radiation of ultraviolet ray which is harmful to DNA sample and human body. As a result, we have found that the above-mentioned purpose can be attained by use of a counter-dye composition containing two kinds of dyes which have contrary electric charges to each other, i. e. a cationic dye and an anionic dye, and have completed the present invention.